Modulation of systemic memory T cell trafficking

ABSTRACT

Methods are provided to specifically modulate the trafficking of systemic memory T cells, particularly CD4+ T cells, without affecting naive T cells or intestinal memory T cells. It is shown that systemic memory T cells, which are characterized as CD45Ra − , and integrin α4β7 − , express high levels of CCR4. Ligands of CCR4, such as TARC or MDC, act as an adhesion trigger, wherein upon CCR4 binding, these cells undergo integrin-dependent arrest to the appropriate vascular receptor(s). This arrest acts to localize the cells at the target site. The methods of the invention manipulate this triggering, and CCR4 mediated chemotaxis, to affect the localization of T cells in targeted tissues. In one embodiment of the invention, the active agent is a CCR4 agonist, that acts to enhance T cell localization. In an alternative embodiment, the agent is an antagonist that blocks CCR4 biological activity. An advantage of the invention is the selectivity for systemic memory T cells, without affecting native T cells or intestinal memory T cells.

CROSS REFERENCE OF RELATED APPLICATIONS

This application is a continuation of prior application No. 09/232,878,filed Jan. 15, 1999.

GOVERNMENT SUPPORT

This work is supported at least in part by grants from the N.I.H.GM37734; and NIH Individual National Research Service Award 1F32Al08930, and was carried out in part in facilities of the Departmentof Veterans Affairs. The government may have certain rights in thisinvention.

INTRODUCTION

Background

During inflammation and immune responses, leukocytes leave the blood andaccumulate at the site of insult. A family of cytokines calledchemokines recruit subsets of leukocytes, and are also involved in acuteand chronic inflammatory processes as well as hematopoiesis. Chemokinesare a subclass of cytokines, which have distinct structural features andbiological effects. Their primary activity is on the chemotaxis ofleukocytes, but they are also reported to have angiogenic andangiostatic effects. All chemokines bind to members of a G-proteincoupled serpentine receptor superfamily that span the leukocyte cellsurface membrane seven times (7-TM). The alpha or CXC chemokines arecharacterized by a single amino acid separating the first 2 cysteines.The beta or CC family of chemokines contain 2 adjacent cysteines. Thehuman genes for the CC chemokines are clustered on chromosome 17q11-q12.

Chemokines are critical in the migration of leukocytes from thecirculatory system to tissues, for example during inflammationprocesses. Most chemokines possess two major binding surfaces: a highaffinity site responsible for specific ligand/receptor interactions anda lower affinity site, also called the heparin-binding orglycosaminoglycan-binding domain, believed to be responsible for theestablishment and presentation of chemokine gradients on the surface ofendothelial cells and within the extracellular matrix. Leukocytes areable to bind to the chemokine gradient through the high affinityreceptor, which then induces remodeling of the leukocyte cytoskeleton,allowing flattening and cellular polarization. Once outside thecirculation, chemokines also guide leukocytes to target tissues.

The chemokine receptor CCR4 was first identified by Power et al. (1995)J. Biol. Chem. 270:19495-19500 (Genbank accession number X85740). It wasoriginally reported that the CC chemokines MIP-1, MCP-1 and RANTES wereable to functionally interact with CCR4. However, recent data hassuggested that this receptor is specific for the chemokines TARC andMDC. CCR4 mRNA is present in basophils, T cells, and monocytes, which isconsistent with the finding that chemokines have been previously shownto exert a diverse range of activities on these cell types, includinghistamine release, chemotaxis, and Ca⁺⁺ mobilization in basophils, andchemotaxis in T cells and monocytes. The expression of CCR4 on Th2 cellshas been reported to be transiently increased following TCR and CD28engagement (D'Ambrosio et al. (1998) J Immunol 161:5111-5). ActivatedTh1 cells also up-regulate CCR4 expression and functional responsivenessto thymus- and activation-regulated chemokine. Analysis of polarizedsubsets of CD8+ T cells reveals a similar pattern of chemokine receptorexpression and modulation of responsiveness.

The chemokine TARC (thymus and activation-regulated chemokine) was firstcloned by Imai et al. (1996) J. Biol. Chem. 271:21514-21521. TARC isexpressed transiently in phytohemagglutinin-stimulated peripheral bloodmononuclear cells and constitutively in thymus. Radiolabeled recombinantTARC bound specifically to T-cell lines and peripheral T cells but notto monocytes or granulocytes, and is able to elicit a chemotacticresponse. Expression of TARC may be upregulated by cytokines known to beproduced by TH2 type T cells.

Macrophage-derived chemokine (MDC) is a recently identified member ofthe CC chemokine family. MDC is not closely related to other chemokines,sharing most similarity with TARC. Northern blot analysis indicates highexpression of MDC in macrophages and in monocyte-derived dendriticcells, but not in monocytes, natural killer cells, or several cell linesof epithelial, endothelial, or fibroblast origin. There are also highexpression levels in thymus and lower expression in lung and spleen.

Both MDC and TARC function as chemoattractants for CCR4 transfectants.Since MDC and TARC are both expressed in the thymus, it has beensuggested that a role for these chemokines may be to attractCCR4-bearing thymocytes in the process of T cell education anddifferentiation (Imai et al. (1998) J Biol Chem 273(3):1764-1768).

Although chemokines are clearly beneficial in wound healing,hematopoiesis, and the clearance of infectious organisms, the continuedexpression of chemokines is associated with chronic inflammation.Therefore, this class of cytokines and/or their receptors are anattractive target for the creation of antagonists that abrogate one ormore chemokine functions. It is envisioned that such antagonists couldserve as a new class of anti-inflammatory drugs.

Relevant Literature

The role of chemokines in leukocyte trafficking is reviewed byBaggiolini (1998) Nature 392:565-8, in which it is suggested thatmigration responses in the complicated trafficking of lymphocytes ofdifferent types and degrees of activation will be mediated bychemokines. The use of small molecules to block chemokines is reviewedby Baggiolini and Moser (1997) J. Exp. Med. 186:1189-1191.

The role of various specific chemokines in lymphocyte homing has beenpreviously described. For example, Campbell et al. (1998) Science,showed that SDF-1 (also called PBSF), 6-C-kine (also called Exodus-2),and MIP-3beta (also called ELC or Exodus-3) induced adhesion of mostcirculating lymphocytes, including most CD4+ T cells; and MIP-3alpha(also called LARC or Exodus-1) triggered adhesion of memory, but notnaive, CD4+ T cells. Tangemann et aL (1998) J. Immunol. 161:6330-7disclose the role of secondary lymphoid-tissue chemokine (SLC), a highendothelial venule (HEV)-associated chemokine, with the homing oflymphocytes to secondary lymphoid organs. Campbell et al. (1998) J. CellBiol 141(4):1053-9 describe the receptor for SLC as CCR7, and that itsligand, SLC, can trigger rapid integrin-dependent arrest of lymphocytesrolling under physiological shear.

The expression of cutaneous lymphocyte antigen (CLA) in human CD4+memory T cell differentiation, and its independent regulation withrespect to cytokine synthesis, is discussed in Teraki and Picker (1997)J Immunol 159(12):6018-29. The skin supports both Th1- andTh2-predominant responses in different settings; and the skin-homingcapability of human memory T cells correlates with and appears to dependon expression of the skin-selective homing receptor CLA. Theidentification of CLA as a specialized form of P-selectin glycoproteinligand-1 is disclosed in Fuhlbrigge et al. (1997) Nature389(6654):978-81. CLA comprises a carbohydrate epitope that facilitatesthe targeting of T cells to inflamed skin, and is defined by both itsreactivity with a unique monoclonal antibody, HECA-452, and its activityas a ligand for E-selectin (reviewed by Butcher and Picker (1996)).

A review of the biology of memory T cells may be found in Dutton et al.(1998) Annu Rev Immunol 16:201-23. Memory cells express a differentpattern of cell surface markers, and they respond in several ways thatare functionally different from those of naive cells. Human memory cellsare CD45RA⁻, CD45RO⁺. In contrast to naive cells, memory cells secrete afull range of T cell cytokines.

SUMMARY OF THE INVENTION

Methods are provided to specifically modulate the trafficking ofsystemic memory T cells, particularly skin-homing cells expressingcutaneous lymphocyte antigen, CLA. Naive T cells and intestinal memory Tcells are not affected by the subject methods. Systemic memory T cellsexpress high levels of the chemokine receptor CCR4, and in response toCCR4 agonists these cells undergo integrin-dependent arrest. In oneembodiment of the invention, naturally occurring CCR4 ligands, whichinclude the chemokines TARC (thymus and activation-regulated chemokine)and MDC (macrophage derived chemokine), are used to specifically attractsystemic memory T cells. Alternative agonists for use as attractantsinclude antibodies and other compounds having specific binding moietiesto CCR4. In another embodiment of the invention, the trafficking ofmemory T cells is prevented by the administration of CCR4 blockingagents; compounds that otherwise prevent the binding of natural CCR4ligands to CCR4; or compounds that prevent expression of, or signalingthrough, CCR4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1E show that skin-homing memory T cells are preferentiallyattracted by TARC and MDC. A comparison is made of unfractionatedlymphocytes from peripheral blood (FIG. 1A) with lymphocytesspecifically attracted to various chemokines (FIG. 1B to FIG. 1E).

FIG. 2 shows the flow cytometry gates used to calculate percentmigration in FIG. 3.

FIG. 3 illustrates the per cent migration of CLA(+)/α4β7(−) andCLA(−)/α4β7(+) memory subpopulations to various chemokines. Migration ofhuman peripheral blood lymphocytes through 5 μm pores, FACs analysis

FIGS. 4A to 4C show differential CCR4 expression in CD4(+) memory T cellsubsets defined by homing receptor expression.

FIGS. 5A to FIG. 5C show TARC-induced rapid adhesion to ICAM-1 enrichedin α4β7(−) memory CD4 cells.

FIGS. 6A to FIG. 6C are plots of CLA(+) memory CD4 cells enriched bybinding to E-selectin transfectants, and α4β7(hi) memory CD4 cellsenriched by binding to MAdCAM-1 transfectants.

FIG. 7 is a graph illustrating that TARC triggers adhesion ofskin-associated but not α4β7(hi) memory CD4 T cells to ICAM-1.

FIG. 8 is a graph demonstrating that TARC triggers rapid adhesion(<1sec) of human lymphocytes rolling on E-selectin under physiologicshear. Adherent lymphocytes accumulate on capillary tube walls undershear only when E-selectin, chemokine and ICAM-1 are all present.

FIGS. 9A and FIG. 9B demonstrate that TARC-induced ICAM-1 adhesion oflymphocytes rolling on E-selectin is extremely rapid.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods are provided to specifically modulate the trafficking ofsystemic memory T cells, particularly CD4⁺ T cells, without affectingnaive T cells and intestinal memory T cells. Systemic memory T cellsexpress high levels of the chemokine receptor CCR4, and in response toCCR4 agonists these cells are triggered to undergo integrin-dependentarrest at a target site. This arrest acts to localize the cells at thetarget site. In some embodiments of the invention, this trigger ismanipulated to modulate the adhesion of these T cells to endothelialcells. The methods of the invention may also modulate the chemotaxis ofthese T cells, which may also control their trafficking and interactionsin systemic sites of inflammation.

In the subject methods, compounds that modulate the triggering activityof CCR4 are administered systemically or locally to alter thetrafficking behavior of the memory T cells. Trafficking, or homing, isused herein to refer to the biological activities and pathways thatcontrol the localization of leukocytes in a mammalian host. Suchtrafficking may be associated with disease, e.g. inflammation, allergicreactions, etc., or may be part of normal biological homeostasis.

Local administration that provides for a prolonged localizedconcentration, which may utilize sustained release implants or othertopical formulation, is of particular interest. In one embodiment of theinvention the trigger modulating compound is an agonist of CCR4, whichacts to enhance the triggering effect. In an alternative embodiment, thetrigger modulating compound blocks CCR4 activity. In vivo uses of themethod are of interest for therapeutic and investigational purposes. Invitro uses are of interest for drug screening, determination ofphysiological pathways, and the like. The subject methods also providefor targeting cells from blood to skin and other systemic sites ofinflammation by expressing CCR4 on the cells to be targeted.

An advantage of the present invention is the selectivity for systemicmemory T cells. Naive T cells are not affected, and therefore much ofthe normal cellular immunity is maintained. Another advantage is theselection for systemic vs. intestinal memory T cells. There are manyconditions that benefit from selective modulation of memory T cells. Forexample, many conditions of chronic inflammation and autoimmunity aremediated by memory T cells, and are improved by preventing such T cellsfrom accumulating target sites by the administration of agents thatblock CCR4 triggering. Such treatment may be prophylactic, e.g. toprevent the onset of disease, or may be used to treat existing disease.

The data presented herein demonstrate that systemic memory T cells,which are characterized as CD45RA⁻, and integrin α4β7⁻, express highlevels of CCR4. Ligands of CCR4 ligands, such as TARC or MDC, act as anadhesion trigger. Upon CCR4 binding, these cells undergointegrin-dependent arrest to the appropriate vascular receptor(s). Ofparticular interest are skin-homing memory T cells, which express highlevels of CLA. The binding to CCR4 triggers arrest of these cells,mediated by the binding of CLA to its counter-receptor, E-selectin.Other systemic memory T cells undergo LFA-1-dependent lymphocyteadhesion to ICAM-1.

For convenience, the nucleic acid sequences (SEQ ID NOS:1, 3, 5) andamino acid sequences (SEQ ID NOS:2, 4, 6) of the native human CCR4, TARCand MDC molecules are provided herein. Unless otherwise specified,references to the molecules made herein are to the moleculescorresponding to these sequences. Nucleic acids having these sequencesmay be used to produce the encoded polypeptides, e.g. to produce antigenfor immunization, for binding studies, to transfect CCR4 into T cells toenhance trafficking, etc.

Systemic memory T cells are characterized according to the cell surfaceexpression of certain known antigens. Typically these cells are positivefor CD4, and lack expression of CD45RA, and integrin α4β7. They arefurther characterized by expression of CCR4. A subset of cells ofinterest are CLA⁺. Verification of the identity of the cells of interestmay be performed by any convenient method, including antibody stainingand analysis by fluorescence detection, ELISA, etc., reversetranscriptase PCR, transcriptional amplification and hybridization tonucleic acid microarrays, etc.

Some memory T cells associated with the skin are known to express CLA,and such cells are of particular interest for treatment with the presentmethods, particularly to modulate the trafficking, or homing of thesecells to cutaneous tissues. Conditions of inflammation-associated orallergic reaction patterns of the skin include atopic dermatitis orinfantile eczema; contact dermatitis, psoriasis, lichen planus;hypersensitivity or destructive responses to infectious agents, etc.Such diseases benefit from the administration of CCR4 agonists. Thetreatment decreases the number of systemic memory T cells at the sitesof inflammation.

Other systemic memory cells are triggered to adhere to endothelialICAM-1, by LFA-1 binding. These adhesion molecules are implicateddirectly in graft rejection, psoriasis, and arthritis. A CCR4 blockingagent that prevents triggering of LFA-1 mediated adhesion is useful inthe inhibition of graft rejection by preventing the accumulation ofmemory T cells at the site of graft implantation; preventing intra-isletinfiltration by T cells to inhibit development of insulin-dependentdiabetes mellitus; blocking infiltration of T cells into the centralnervous system to treat multiple sclerosis and other demyelinatingdiseases; blocking the accumulation of T cells in the synovial joints ofpatients suffering from rheumatoid arthritis; accumulation of memory Tcells to influence immune responsiveness, and the like.

CCR4 agonists are useful in enhancing the immune reaction at a targetedsite. For example, in burn patients it may be desirable toprophylactically increase the memory T cell population at the burnsites. Other infections, particularly localized infections, may betreated this way, including, without limitation, human herpes virusesincluding herpes simplex viruses (HSV) types 1 and 2, Epstein Barr virus(EBV), cytomegalovirus (CMV), varicella zoster virus (VZV) and humanherpes virus 6 (HHV-6), particularly infections of the mouth andgenitals, hepatitis B virus (HBV) and hepatitis C virus (HCC) infectionsof the liver, etc.

CCR4 modulating agents are molecules that specifically act as an agonistto enhance CCR4 biological activity; or that act as antagonists thatblock CCR4 biological activity, for example the interaction between CCR4and its ligands. Often such agents interact with the extracellularbinding domain or transmembrane domain of CCR4 protein, and may activatethe molecule through the ligand binding site, block the ligand bindingsite, conformationally alter the receptor, etc. Usually the bindingaffinity of the blocking agent will be at least about 100 μM. Preferablythe blocking agent will be substantially unreactive with relatedmolecules to CCR4, such as CCR1, CCR2, CCR3, CCR5, etc. and othermembers of the seven transmembrane domain superfamily. Blocking agentsdo not activate CCR4 triggering of adhesion. Agonists may activate thetriggering activity, enhance chemotaxis activity, or enhance thetriggering activity of other ligands. It will be understood by one ofskill in the art that the following discussions of cross-reactivity andcompetition between different molecules is intended to refer tomolecules having the same species of origin, e.g. human CCR4 binds humanTARC and MDC, etc.

Candidate modulating agents are screened for their ability to meet thiscriteria. Assays to determine affinity and specificity of binding areknown in the art, including competitive and non-competitive assays.Assays of interest include ELISA, RIA, flow cytometry, etc. Bindingassays may use purified or semi-purified CCR4 protein, or alternativelymay use native memory T cells that express CCR4, or other cells, e.g.cells transfected with an expression construct for CCR4; membranes fromthese cells; etc. As an example of a binding assay, purified CCR4protein is bound to an insoluble support, e.g. microtiter plate,magnetic beads, etc. The candidate modulating agent and soluble, labeledTARC or MDC are added to the cells, and the unbound components are thenwashed off. The ability of the modulating agent to compete with TARC andMDC for CCR4 binding is determined by quantitation of bound, labeledTARC or MDC. Confirmation that the blocking agent does not cross-reactwith other chemokine receptors may be performed with a similar assay,substituting CCR1, CCR2, etc. for CCR4. Suitable molecules will have atleast about 10² less binding to other chemokine receptors than to CCR4,more usually at least about 10³ less binding.

A number of screening assays are available for blocking agents. Thecomponents of such assays will typically include CCR4 protein; andoptionally a CCR4 activating agent, e.g. TARC, MDC, etc. The assaymixture will also comprise a candidate pharmacological agent. Generallya plurality of assay mixtures are run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e. at zero concentration or below the level ofdetection.

Conveniently, in these assays one or more of the molecules will bejoined to a label, where the label can directly or indirectly provide adetectable signal. Various labels include radioisotopes, fluorescers,chemiluminescers, enzymes, specific binding molecules, particles, e.g.magnetic particles, and the like. Specific binding molecules includepairs, such as biotin and streptavidin, digoxin and antidigoxin etc. Forthe specific binding members, the complementary memberwould normally belabeled with a molecule which provides for detection, in accordance withknown procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal protein-DNAbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.may be used.

A functional assay that detects T cell adhesion triggering may be usedfor confirmation. For example, a population of systemic memory T cellsmay be stimulated with TARC or MDC, in the presence or absence of thecandidate modulating agent. An agent that blocks CCR4 triggering willcause a decrease in the T cell adhesion to the appropriate endothelialcell molecule, e.g. LFA-1 or E-selectin, as measured by the assaysdescribed in the examples provided herein, etc. An agent that is a CCR4agonist will increase adhesion of the T cells to such an endothelialcell molecule, either in the absence, or in the presence of CCR4ligands.

CCR4 modulating agents are peptides, small organic molecules,peptidomimetics, soluble T cell receptors, antibodies, or the like.Antibodies are an exemplary modulating agent. Antibodies may bepolyclonal or monoclonal; intact or truncated, e.g. F(ab′)₂, Fab, Fv;xenogeneic, allogeneic, syngeneic, or modified forms thereof, e.g.humanized, chimeric, etc.

In many cases, the modulating agent will be an oligopeptide, e.g.antibody or fragment thereof, etc., but other molecules that providerelatively high specificity and affinity may also be employed.Combinatorial libraries provide compounds other than oligopeptides thathave the necessary binding characteristics. Generally, the affinity willbe at least about 10⁻⁶, more usually about 10⁻⁸ M, i.e. bindingaffinities normally observed with specific monoclonal antibodies.

Candidate agents also encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl, sulfhydryl orcarboxyl group, preferably at least two of the functional chemicalgroups. The candidate agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Candidateagents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

Suitable antibodies for use as blocking agents are obtained byimmunizing a host animal with peptides comprising all or a portion ofCCR4 protein. Suitable host animals include mouse, rat sheep, goat,hamster, rabbit, etc. The origin of the protein immunogen may be mouse,human, rat, monkey etc. The host animal will generally be a differentspecies than the immunogen, e.g. mouse CCR4 used to immunize hamsters,human CCR4 to immunize mice, etc.

The immunogen may comprise the complete protein, or fragments andderivatives thereof. Preferred immunogens comprise all or a part of theextracellular domain of human CCR4, where these residues containpost-translation modifications, such as glycosylation, found on thenative protein. Immunogens comprising the extracellular domain areproduced in a variety of ways known in the art, e.g. expression ofcloned genes using conventional recombinant methods, isolation from Tcells, sorted cell populations expressing high levels of CCR4, etc.Where expression of a recombinant or modified protein is desired, avector encoding the desired portion of CCR4 will be used. Generally, anexpression vector will be designed so that the extracellular domain ofthe CCR4 molecule is on the surface of a transfected cell, oralternatively, the extracellular domain is secreted from the cell.

Monoclonal antibodies are produced by conventional techniques.Generally, the spleen and/or lymph nodes of an immunized host animalprovide a source of plasma cells. The plasma cells are immortalized byfusion with myeloma cells to produce hybridoma cells. Culturesupernatant from individual hybridomas is screened using standardtechniques to identify those producing antibodies with the desiredspecificity. Suitable animals for production of monoclonal antibodies tothe human protein include mouse, rat, hamster, etc. To raise antibodiesagainst the mouse protein, the animal will generally be a hamster,guinea pig, rabbit, etc. The antibody may be purified from the hybridomacell supernatants or ascites fluid by conventional techniques, e.g.affinity chromatography using CCR4 bound to an insoluble support,protein A sepharose, etc.

The antibody may be produced as a single chain, instead of the normalmultimeric structure. Single chain antibodies are described in Jostetal. (1994) J.B.C. 269:26267-73, and others. DNA sequences encoding thevariable region of the heavy chain and the variable region of the lightchain are ligated to a spacer encoding at least about 4 amino acids ofsmall neutral amino acids, including glycine and/or serine. The proteinencoded by this fusion allows assembly of a functional variable regionthat retains the specificity and affinity of the original antibody.

For in vivo use, particularly for injection into humans, it is desirableto decrease the antigenicity of the blocking agent. An immune responseof a recipient against the blocking agent will potentially decrease theperiod of time that the therapy is effective. Methods of humanizingantibodies are known in the art. The humanized antibody may be theproduct of an animal having transgenic human immunoglobulin constantregion genes (see for example International Patent Applications WO90/10077 and WO 90/04036). Alternatively, the antibody of interest maybe engineered by recombinant DNA techniques to substitute the CH1, CH2,CH3, hinge domains, and/or the framework domain with the correspondinghuman sequence (see WO 92/02190).

The use of Ig cDNA for construction of chimeric immunoglobulin genes isknown in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987) J.Immunol. 139:3521). mRNA is isolated from a hybridoma or other cellproducing the antibody and used to produce cDNA. The cDNA of interestmay be amplified by the polymerase chain reaction using specific primers(U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library ismade and screened to isolate the sequence of interest. The DNA sequenceencoding the variable region of the antibody is then fused to humanconstant region sequences. The sequences of human constant regions genesmay be found in Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, N.I.H. publication no. 91-3242. Human C regiongenes are readily available from known clones. The choice of isotypewill be guided by the desired effector functions, such as complementfixation, or activity in antibody-dependent cellular cytotoxicity.Preferred isotypes are IgG1, IgG3 and IgG4. Either of the human lightchain constant regions, kappa or lambda, may be used. The chimeric,humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g. by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g. SV-40 earlypromoter, (Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcomavirus LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murineleukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native Igpromoters, etc.

The formulation of CCR4 modulating agent is administered at a doseeffective to alter the accumulation of systemic memory T cells at thetargeted site, e.g. a site of inflammation, cutaneous tissue, etc. Thesubject invention is useful in any species, such as primate,particularly human, domestic animals, e.g. murine, bovine, equine,canine, feline, ovine, porcine, etc., and any of these species may findapplication as a source of antibodies.

The antibodies or other binding molecules used in the method of thepresent invention are preferably administered to individuals in a mannerthat will maximize the likelihood of the antibody or otherepitope-binding molecule reaching the targeted cell, binding to it, andthereby modulating the interaction of chemokine and receptor. The dosefor individuals of different species and for different diseases isdetermined by measuring the effect of the modulating agent on thelessening of parameters that are indicative of the disease beingtreated.

The CCR4 modulating agent can be given by various conventionaladministration routes, e.g. oral, rectal, intravenous, subcutaneous,intraperitoneal, transdermal, etc. Formulations of the CCR4 modulatingagent are administered to a host affected by an immune disordercharacterized by undesirable numbers of systemic memory T cells at atarget site. The blocking agents of the invention are administered at adosage that reduces the number of systemic memory T cells at a targetsite. The blocking agents of the present invention are administered at adosage that reduces the numbers of memory T cells, thereby reducing Tcell mediated immune activation, particularly chronic inflammation,while minimizing any side-effects. The CCR4 agonists enhance immunereactivity, for example as prophylaxis during trauma to the skin, e.g.burn victims and the like; as a treatment for infection; etc. It iscontemplated that the composition will be obtained and used under theguidance of a physician for in vivo use.

Various methods for administration may be employed. The formulation maybe given orally, by inhalation, or may be injected, e.g. intravascular,subcutaneous, intraperitoneal, intramuscular, etc. The dosage of thetherapeutic formulation will vary widely, depending upon the nature ofthe disease, the frequency of administration, the manner ofadministration, the clearance of the agent from the host, and the like.The initial dose may be larger, followed by smaller maintenance doses.The dose may be administered as infrequently as weekly or biweekly, orfractionated into smaller doses and administered daily, semi-weekly,etc. to maintain an effective dosage level.

The CCR4 modulating agents of the invention can be incorporated into avariety of formulations for therapeutic administration. Moreparticularly, the agents can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, granules, ointments, solutions, suppositories,injections, inhalants, gels, microspheres, and aerosols. As such,administration of the CCR4 modulating agents can be achieved in variousways, including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intracheal, etc., administration. The agentsmay be systemic after administration or, preferably, are localized bythe use of an implant that acts to retain the active dose at the site ofimplantation.

In pharmaceutical dosage forms, the CCR4 modulating agents may beadministered in the form of their pharmaceutically acceptable salts, orthey may also be used alone or in appropriate association, as well as incombination with other pharmaceutically active compounds. The followingmethods and excipients are merely exemplary and are in no way limiting.

For oral preparations, the CCR4 modulating agents can be used alone orin combination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The CCR4 modulating agents complexes can be formulated into preparationsfor injections by dissolving, suspending or emulsifying them in anaqueous or nonaqueous solvent, such as vegetable or other similar oils,synthetic aliphatic acid glycerides, esters of higher aliphatic acids orpropylene glycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

The CCR4 modulating agents complexes can be utilized in aerosolformulation to be administered via inhalation. The compounds of thepresent invention can be formulated into pressurized acceptablepropellants such as dichlorodifluoromethane, propane, nitrogen and thelike.

Furthermore, the CCR4 modulating agents complexes can be made intosuppositories by mixing with a variety of bases such as emulsifyingbases or water-soluble bases. The CCR4 modulating agents of the presentinvention can be administered rectally via a suppository. Thesuppository can include vehicles such as cocoa butter, carbowaxes andpolyethylene glycols, which melt at body temperature, yet are solidifiedat room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsof the present invention. Similarly, unit dosage forms for injection orintravenous administration may comprise the compound of the presentinvention in a composition as a solution in sterile water, normal salineor another pharmaceutically acceptable carrier.

Implants for sustained release formulations are well-known in the art.Implants are formulated as microspheres, slabs, etc. with biodegradableor non-biodegradable polymers. For example, polymers of lactic acidand/or glycolic acid form an erodible polymer that is well-tolerated bythe host. The implant containing CCR4 modulating agents is placed inproximity to the site of action, so that the local concentration ofactive agent is increased relative to the rest of the body.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of CCR4modulating agents of the present invention calculated in an amountsufficient to produce the desired effect in association with apharmaceutically acceptable diluent, carrier or vehicle. Thespecifications for the novel unit dosage forms of the present inventiondepend on the particular complex employed and the effect to be achieved,and the pharmacodynamics associated with each complex in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

The compositions of the invention may also contain other therapeuticallyactive agents. Of particular interest are combinations with other agentscapable of additive or synergistic effect in achieving a therapeuticresult, e.g. where a different or complementary pathway is affected byeach of the active agents. The combined use of CCR4 modulating agent andother agents has the advantage that the required dosages for theindividual drugs may be lower, and the onset and duration of effect ofthe different drugs complementary. In the combined therapy the differentactive agents may be delivered together or separately, andsimultaneously or at different times within the day.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Some of the specificcomplexes are more potent than others. Preferred dosages for a givenagent are readily determinable by those of skill in the art by a varietyof means. A preferred means is to measure the physiological potency of agiven compound.

EXPERIMENTAL

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,constructs, and reagents described, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which scope will be determined by thelanguage in the claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “amouse” includes a plurality of such mice and reference to “the cytokine”includes reference to one or more cytokines and equivalents thereofknown to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

All publications mentioned herein are incorporated herein by referencefor all relevant purposes, e.g., the purpose of describing anddisclosing, for example, the cell lines, constructs, and methodologiesthat are described in the publications which might be used in connectionwith the presently described invention. The publications discussed aboveand throughout the text are provided solely for their disclosure priorto the filing date of the present application. Nothing herein is to beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

Example 1

To identify circulating lymphocyte subsets responsive to the CCR4ligands, the phenotype of peripheral blood lymphocytes (PBL) migratingto its chemokine ligands TARC and MDC were analyzed in a standardtranswell chemotaxis assay (Campbell et al. (1996) J. Cell Biol. 134,255-266; Campbell et al. (1998) J. Cell Biol. 141, 1053-1059). Flowcytometric analyses were carried out to identify natural killer (CD56⁺,CD16⁺, CD3⁻) cells, B cells (CD19⁺), and naive (CD45RA⁺) and memory(CD45RA⁻) CD4⁺and CD8⁺ T cells. Because of interest in determining therole of chemokines in regional lymphocyte trafficking, memory T cellsub-populations were also analyzed, as defined by expression of theintegrin α4β7, which is the lymphocyte receptor for the mucosaladdressin cell adhesion molecule (MAdCAM-1). α4β7^(hi) memorylymphocytes bind to vascular MAdCAM-1, traffic to intestinal sites, andcarry memory for intestinal antigens; whereas α4β7⁻ cells embody memoryfor systemic immune responses (Santamaria-Babi et al. (1995) J. Exp.Med. 181, 1935-1940). As illustrated in FIG. 1, all lymphocyte subsetsexcept natural killer cells responded well to MIP3β(a ligand for CCR7),and all subsets migrated to SDF-1 (a ligand for CXCR4). In contrast,α4β7⁻ (non-intestinal, systemic) memory CD4⁺ T cells were thepredominant population recruited by TARC and by MDC; and α4β7⁻ memoryphenotype CD8⁺ cells were also enriched among lymphocytes responding tothese chemokines.

Migration of human peripheral blood lymphocytes through 5 μm pores andFACs analysis of migrated cells was performed as described (Campbell etal. (1998), supra.) Lymphocyte subsets were defined as follows: NaturalKiller=CD56(+), CD16(+), CD3(−); B Cell=CD19(+); CD8(+) T Cell(naive)=TCRαβ(+), CD8(hi), CD27(+), CD45RA(hi); CD8(+) T Cell (α4β7(+)Memory)=CD8(hi), CD56(−), CD45RA(neg), α4β7(+); CD8(+) T Cell (α4β7(−)Memory)=CD8(hi), CD56(−), CD45RA(neg), α4β7(−); CD4(+) T Cell(naive)=CD4(+), CD45RA(+); CD4(+) T Cell (α4β7(+) Memory)=CD4(+),CD45RA(−), α4β7(+); CD4(+) T Cell (α4β7(−) Memory)=CD4(+), CD45RA(−),α4β7(−). Mean and SD from 3 individual healthy donors is shown, with4-16 replicate wells per chemokine per donor (4 for SDF-1α and MIP-3β;16 for TARC and MDC), and 24 wells for control lacking chemokines. Tocalculate specific migration, the mean number of cells/well thatmigrated into the bottom well in the absence of chemokine (background)was subtracted from the total number of cells/well that migrated tochemokine in parallel wells. To calculate the % representation of eachlymphocyte subtype in the migrated populations, the number ofspecifically migrated cells/well belonging to each subtype was dividedby the total number of specifically migrated cells/well.

The selective activity of the CCR4 ligands on circulating α4β7⁻ memorycells suggests a role in tissue-selective lymphocyte recruitment fromthe blood. To explore this possibility further, the responses of the twobest-characterized, antigenically-defined tissue-targeted memory T cellsubsets were compared. These subsets were intestinal (α4β7^(hi)) memoryCD4⁺ cells, and skin homing memory CD4⁺ T cells defined by the cutaneouslymphocyte antigen, CLA. FIG. 2 shows the profile of peripheral bloodlymphocytes from a typical healthy donor. CLA vs α4β7 staining ofCD4(+)/CD45RA(−) memory lymphocytes from peripheral blood. Gates aredrawn around the skin associated CLA(+)/α4β7(−) and intestinalCLA(−)/α4β7(+) memory subpopulations.

CLA is a sialyl Lewis^(x)-related carbohydrate-dependent T cell epitopethat identifies cutaneous memory cells and functions as a T cell homingreceptor for skin. As shown in FIG. 3, TARC and MDC attract these skinhoming memory cells extremely well, whereas intestinal α4β7^(hi) memoryT cells respond poorly. α4β7⁻ CLA⁻ T cells also migrated significantly,although consistently less well than the CLA⁺ population, suggestingthat responsiveness of cutaneous memory lymphocytes for CCR4 ligands maybe shared with a subset of other systemic memory cells.

The calculation of migrated cells and calculation of % migration in FIG.3 were performed as described in Campbell et al. (1998), supra. Briefly,migration assays were carried out in RPMI-1640 with 0.5% BSA using24-well plate tissue culture inserts (Costar Corp., Cambridge, Mass.)with 5 μm pore polycarbonate filters. 5×10⁵ cells were placed in theupper chamber in 100 μl, 600 μl of chemokine dilution in the lower well;and migration carried out for 90 min at 37° C. To calculate per centmigration, the number of cells of each subtype was determined for thestarting population of each chemotactic assay. Next, the number of cellsbelonging to the same subtype was determined for the migratedpopulation, and the per cent migration determined from these twonumbers. Mean and SD from 6 individual experiments is shown (wells perexperiment described in FIG. 1). Mean background migration of 1.07% forCLA(−)/α4β7(+) cells and 2.74% for CLA(+)/α4β7(−) cells has beensubtracted. The difference in % migration between CLA(+)/α4β7(−) andCLA(−)/α4β7(+) is highly significant by the Mann-Whitney rank order testfor TARC and MDC (p<0.01).

Consistent with these chemotactic responses, immunofluorescence stainingconfirmed differential expression of CCR4 on skin-associated vs.intestinal memory cells. As illustrated in FIG. 4, most CLA⁺ memory CD4cells express very high levels of CCR4 (>95% positive; >70% over 100mean fluorescence units), whereas most α4β7^(hi) memory cells are weakor negative (<30% positive; 90% <100 mean fluorescence units). Thesedata demonstrate that CCR4 and its ligands mediate preferentialrecruitment of α4β7⁻ systemic memory T cells, especially the skin homingpopulation.

In FIG. 4, peripheral blood memory CD4(+) subsets were defined by fourcolor flow cytometry as follows: CLA⁺ (left panel)=CD4(+), CLA(+),α4β7(−); CLA-/(α4β7⁻ (center panel)=CD4(+), CLA(−), α4p7(−); α4β7^(hi)(right panel)=CD4(+), CD45RA(−), α4β7(hi). Expression of CCR4 wasdetermined by MAb 1 Gl produced against CCR4-transfected mouse L1/2cells essentially as described in Qin et al. (1996) Eur. J. Immunol.26:640-647 and shown to specifically stain CCR4-expressing L1/2 cellsbut not control transfectants expressing other chemokine and relatedorphan receptors. Parallel staining with isotype-matched control IgG1was negative for these subsets. Combined staining of three individualhealthy donors is shown. Reagents: Chemokines were titered and used atthe optimal concentrations for chemotaxis of unfractionated lymphocytesas follows: TARC, MDC and SDF-1α=100 nM, MIP-3β=1 μM. Synthetic humanTARC and SDF-1α were obtained from Gryphon Sciences (South SanFrancisco, Calif.). Recombinant human MDC was obtained from Amgen(Boulder Colo.) and MIP-3β was purchased from PeproTech EC, Ltd. (RockyHill, N.J.). Directly conjugated antibodies used for FACs analysis wereobtained from PharMingen, Inc. (San Diego, Calif.) unless otherwiseindicated. FITC conjugated anti- CD3 (clone UCHT1), CD45RA (cloneHI100), CLA (clone HECA 452, prepared by Butcher lab staff); PEconjugated anti-CD56 (clone B159), CD19 (clone B43), CD27 (M-T271), α4β7(clone ACT-1, LeukoSite, Inc.); Biotinylated anti-CD45RA (clone HI100),CD16 (clone3G8), CD8 (clone RPA-T8); APC conjugated anti-CD4 (cloneRPA-T4), CD8 (clone RPA-T8), CD56 (clone B159), TCRαβ (cloneT10B9.1A-31). The second stage reagent used for all biotinylatedantibodies was streptavidin-conjugated PerCP (Beckton Dickinson, SanJose, Calif.).

Chemokines may control lymphocyte trafficking not only throughstimulation of chemotaxis, but also by triggering rapidintegrin-dependent adhesion and arrest of lymphocytes on endothelium(Butcher (1991) Cell 67:1033-1036; Butcher & Picker (1996) Science272:60-66). Consistent with this hypothesis, certain chemokines cantrigger rapid arrest of lymphocytes under physiologic shear; and it iswell documented that some chemokines can be expressed and/or presentedby endothelial cells at sites of lymphocyte extravasation.

We have found that TARC can be expressed by activated endothelium. TARC,but not MDC, message was readily detected by Northern blot analyses inRNA from endotoxin- and cytokine-stimulated human umbilical veinendothelial cells; and TARC message was observed in primary cultures ofenriched human tonsil HEV cells. Thus TARC can be expressed byendothelium, and therefore might be available for regulation of vascularinteractions by lymphocytes.

The differential expression of CCR4 could contribute to lymphocyterecognition of cutaneous endothelium if TARC were displayed by venulesin the skin. Immunohistological analyses of biopsies of chronicallyinflamed skin from patients with a variety of dermatologic disordersrevealed reactivity of anti-TARC monoclonal antibody (MAb) with venulesassociated with lymphocyte recruitment, including (but not limited to)most E-selectin-expressing venules.

Frozen sections were fixed 10 min at RT in 4% paraformaldehyde inphosphate buffered saline (PBS). After washing in PBS, standardimmunoperoxidase staining was performed as described by Picker etal.(1991) Nature 349:796-800). MAb LS142-2D8 was produced against synthetichuman TARC by standard techniques, and selected by ELISA for reactivitywith TARC but not MDC or other chemokines.

Anti-TARC MAb (LS142-2D8) stains endothelial cells lining venulesassociated with lymphocyte infiltration in a case of psoriasis.Juxta-epidermal vessels were stained with anti-TARC but not with controlIgG1 MAb. Venules in the dermis were also positive. In contrast,anti-TARC MAb did not stain a MAdCAM-1-positive venule in the coloniclamina propria. TARC reactivity was also observed in chronicallyinflamed skin in biopsies of lichen planus, atopic dermatitis andnon-specific chronic inflammation.

Venules in minimally inflamed areas of the dermis were often positive aswell, if less intensely and consistently. Reactivity was also observedon many high endothelial venules in inflamed tonsils. These vessels,thought to mediate recruitment of subsets of memory as well as naivelymphocytes, may express several chemokines to support interactions ofdiverse B and T cell subsets. In contrast, venules involved recruitmentto gastrointestinal lamina propria (identified by MAdCAM-1 staining inbiopsies of small and large intestine, stomach, and normal and inflamedcolon) were usually negative; and reactivity when observed was focal andweak. Parallel immunohistological studies of macaque skin revealedscattered constitutive anti-TARC reactivity of venules, but with moreextensive reactivity in experimentally induced delayed typehypersensitivity reactions, generated as described in Silber et al.(1994) J. Clin. Invest. 93:1554-1563. The results suggest that TARC isexpressed in a regionally selective fashion by activated endothelium,and is well positioned to help control the vascular adhesion and arrestof circulating CCR4⁺ lymphocytes on inflamed endothelium in skin.

It was then determined whether TARC could trigger rapid integrinactivation and integrin-dependent arrest of circulating lymphocytes. Ininitial assays of rapid LFA-1-dependent lymphocyte adhesion toimmobilized ICAM-1 in vitro, the CCR4 ligand TARC failed to induceadhesion of whole PBL that was detectable above background (Campbell etal. (1998) Science 279:381-384). However, purified CD4⁺ lymphocytesdisplayed a significant response to TARC; and isolated α4β7⁻ memory CD4⁺lymphocytes demonstrated robust adhesion with up to 50% of input cellsbinding firmly to ICAM-1 within 2 mins. after chemokine addition (FIG.5).

Moreover, TARC differentially triggered adhesion of skin vs. intestinalT cells. CLA⁺ and α4β7^(hi) memory cells were enriched by bindingisolated CD4⁺ T cells to transfected fibroblasts expressing the CLAcounter-receptor E-selectin, or the α4β7 receptor MAdCAM-1, followed byelution with cation chelating buffer as previously described. Asillustrated in FIG. 5, although both populations responded equally wellto SDF-1, TARC selectively induced adhesion of the E-selectin-enrichedCLA⁺ population to ICAM-1. Interestingly, although a subset of α4β7^(hi)T cells do express CCR4, and α4β7^(hi) cells do migrate above backgorundto TARC (albeit very poorly), they display little or no rapid adhesionto ICAM-1: the low levels of CCR4 on the positive subset of intestinalmemory cells (˜7 fold lower median fluorescence than CLA+ T cells) maybe insufficient to generate a robust proadhesive response, whichtypically requires engagement of large numbers of receptors.

The static rapid adhesion assays shown in FIG. 5 were carried out asdescribed in Campbell et al. (1998) Science 279:381-384. Briefly, humanlymphocytes were allowed to settle in multiwell glass slides coated withICAM-1 to a density of ˜1000 sites per square micrometer. After cellsettling, the indicated chemokines were added to a final concentrationof 1 μM. The slides were washed at the indicated times to removenonadherent cells, and bound cells were then fixed. Adherent cells inthe microscopic field proximal to the site of chemoattractant additionwere counted. Error bars indicate range of duplicate wells. Results arerepresentative of experiments with two different donors. Testmononuclear cells were purified by density separation on ficoll, andeither depleted of monoytes by incubation on plastic for direct use asunfractionated lymphocytes (left panel); or positively selected for CD4expression (center and right panels). CD4(+) cells were purified fromPBL with the use of anti-CD4 Dynabeads and the DETACH-a-BEAD system(Dynal, Lake Success, N.Y.) and used directly (center panel); ordepleted of naive and α4β7(+) memory cells (right panel), by incubationwith mouse anti-human CD45RA and mouse anti-human α4β7 (unconjugatedversions of the same MAbs used in FIG. 1), followed by microbeads coatedwith anti-mouse immunoglobulin, and magnetic depletion (Miltenyi Biotec,Auburn, Calif.). A portion of each processed cell population was stainedwith directly conjugated antibodies and analyzed by flow cytometry toascertain purity. The CD4(+) population was 99% pure, and theCD4(+)/CD45RA(−)/α4β7(−) population was 98% CD45RO(+) and 22% CLA(+).FIG. 6. Purified CD4(+) T lymphocytes were incubated on MAdCAM-1 (CHOcells transfected with murine MAdCAM-1) or E-selectin (CHO-K cellstransfected with human E-selectin, PDL, Inc.) in a T-175 at 37° C. for30 min with occasional agitation, non-bound cells were washed away withwarm complete medium, and bound cells were recovered by elution withdivalent-cation-free HBSS for E-selectin or 0.5mM EDTA for MAdCAM-1.Approximately 70% of the E-selectin-adherent population was CLA⁺, andonly 6% were α4β7^(hi) (with the remainder naive and α4β7⁻ memorycells); where cells enriched on MAdCAM-1 consisted of ˜60% α4β7^(hi)cells and only ˜5% CLA⁺ cells. Data presented is representative of twoindividual experiments with different donors.

Enriched populations from above were tested for chemokine-inducedadhesion to ICAM-1, shown in FIG. 7. Adhesion was assessed 2 min afterchemokine addition. Background adhesion of 11% of input forE-selectin-enriched cells and 10% of input for MAdCAM-1-enriched cellswas subtracted. Data shown are representative of 2 experiments (withdifferent donors) with 2 duplicate wells for each data point. Error barsindicate range of duplicates.

Triggered adhesion of lymphocytes to endothelium in vivo must occurwithin seconds and under conditions of strong shear stress at thevascular wall. In inflamed skin, the vascular CLA receptor E-selectin isthought to mediate initial cell contact (tethering) and support rolling,and β2 integrin ligands mediate arrest, with a variable contribution ofα4β1 integrin and VCAM-1 (Butcher & Picker (1996), supra.) Therefore, todetermine whether the CCR4 ligand TARC could mediateactivation-dependent arrest of lymphocytes rolling under physiologicconditions, we coated capillary tubes with E-selectin in combinationwith TARC and/or ICAM-1, and passed PBL through the tube at a wall shearstress of 2 dynes/cm². As illustrated in FIG. 8, many rollinglymphocytes came to a rapid stop. Arrest occurred within less than 1second after initial rolling for most cells (FIGS. 9A and 9B), andrequired the combined presence of all three components: lymphocyterolling was unperturbed in the absence of ICAM-1 or TARC, but arrest wasdramatically less efficient (FIG. 8). We conclude that TARC can combinewith vascular E-selectin and integrin ligands in a multi-step adhesioncascade in which both CLA/E-selectin and CCR4/TARC contribute to theselective arrest of skin homing T cells.

The adhesion assays under shear, shown in FIG. 8, were performed asdescribed above. Briefly, cells at 2×10⁶/ml were a passed through acapillary tube (1.025 mm inner diameter, Drummond, Broomall, Pa.) at1250 μl/min (controlled by a Harvard 33 syringe pump, Harvard Apparatus,South Natick, Mass.), which generates a wall shear stress of ˜2.0dynes/cm². Adherent cells were counted in 10 fields (fields recorded at30 sec intervals) between 6 and 11 min after the start of the assay.Combined data for 4 different healthy donors (10 fields each) is shown.Error bars indicate SD. The difference in cell accumulation betweentubes coated with E-selectin, TARC and ICAM-1 and tubes missing eitherTARC or ICAM-1 was highly significant for the four donors by theMann-Whitney rank order test (p<0.03). Human E-selectin was purifiedfrom E-selectin-transfected L1/2 cells on an affinity column ofmonoclonal antibody E8.16-3 (PDL, Inc.) conjugated to Sepharose, usingthe tissue lysis procedure previously described (Honda et al. J.Immunol. (1994) 152:4026-4035). ICAM-1 was prepared from mouse spleenand lymph nodes as described in Campbell et al. (1998), supra.Detergent-solubilized adhesion molecules (ICAM-1 and E-selectin) andchemokines were coated on the inner surfaces of the tubes as previouslydescribed (infra.)

FIG. 9 shows the plots depicting behavior of individual cellsinteracting with the coated areas of capillary tubes, as describedabove. Each line represents a cell that began interaction with thecoated adhesion molecules within the field of observation. The slope ofthe line is proportional to the velocity of the cell. The behavior of 5representative cells is shown for each assay. The y axis indicates thelength of the microscopic field (220 μm), with zero as the entry intothe field and 220 as the downstream exit from the field. The depictedtime at which the cell enters the field is arbitrary. Capillary tubeswere coated with a combination of E-selectin and ICAM-1 plus eithermedium alone (FIG. 9A) or TARC (FIG. 9B). The mean velocity of tumblingcells was 327 μm/sec (+88 SD) under these conditions, determined for 10randomly chosen cells on uncoated areas of the capillary. The meanvelocity of rolling cells was 5.1 μm/sec (+1.9 SD), determined for 10randomly chosen cells on E-selectin+ICAM-1 (without TARC). Thedeceleration time was defined as the time elapsed between the point atwhich the cell's speed dropped two standard deviations below the meantumbling velocity (327−176=151 μm/sec) and the point at which the cellcame to a complete stop. The mean deceleration time was determined for13 cells that remained arrested for >1 min, and was 0.87 sec (+0.92 SD).The longest deceleration was 2.51 sec for a cell that initiallyarrested, then rolled another cell length before coming to completearrest. The fastest deceleration time was <0.03 sec, the duration of aVHS video frame, which was observed for 3 of the 13 cells.

Recent reports have suggested a role for CCR4 and its ligands,especially MDC, in selective chemotaxis of in vitro-polarized Th2 Tlymphocytes. However, CLA⁺ T cells are not enriched in Th2 cells. Infact, upon short term stimulation with general activating stimuli,essentially identical fractions of blood memory CLA⁺ and CLA⁻CD4 cellsdisplay Th1 vs. Th2 cytokine profiles.

The above data demonstrates that, on circulating memory lymphocytes,CCR4 functions as a chemoattractant “homing receptor” for skin andpotentially other non-intestinal tissues, independent of cytokinecommitment patterns. Its differential expression and function on fullypolarized Th2 vs. Th1 cells in vitro may imply a parallel role for therelatively infrequent effector Th2 cells that enter the blood, or may bemore relevant to the behavior of activated effector T cells withinextravascular sites of inflammation.

In conclusion, TARC and its lymphocyte receptor CCR4 are involved in thehoming of circulating memory T cells, and in their interactions withvascular endothelium in cutaneous sites of inflammation. They providethe first evidence for chemokine involvement in tissue-selectivelymphocyte-endothelial recognition, and suggest that CCR4 and itsligands play a fundamental role in the regional targeting of memory Tcells, and thus in the functional segregation of intestinal vs.systemic, especially cutaneous, immune responses.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method of screening comprising: assessing an effect of an agent ona cell expressing CCR4 in the presence of TARC or MDC.
 2. The method ofclaim 1, wherein the cell expressing CCR4 is a T cell.
 3. The method ofclaim 1, wherein the effect is binding of TARC or MDC to the cell. 4.The method of claim 1, wherein the effect is adhesion of the cell.
 5. Amethod of modulating the trafficking of CCR4⁺ leukocytes, comprising:modulating the interaction between CCR4 and MDC or TARC.
 6. The methodof claim 5, wherein the leukocyte is a lymphocyte.
 7. The method ofclaim 6, wherein the lymphocyte is a T cell.
 8. The method of claim 7,wherein the T cell is CD4⁺.
 9. The method of claim 7, wherein the T cellis CD8⁺.
 10. The method of claim 5, wherein the modulated interactionaffects adhesion of the CCR4⁺ leukocytes.
 11. The method of claim 5,wherein the modulated interaction affects chemotaxis of the CCR4³⁰leukocytes.
 12. The method of claim 5, wherein the interaction ismodulated by CCR4 agonist activity.
 13. The method of claim 5, whereinthe interaction is modulated by CCR4 antagonist activity.
 14. A methodof treating inflammatory disease, comprising: interfering with theinteraction between CCR4 and TARC or MDC.
 15. The method of claim 14,wherein the inflammatory disease is an inflammatory skin disease. 16.The method of claim 14, wherein the method comprises administering to apatient an effective amount of a CCR4 antagonist.
 17. The method ofclaim 16, wherein the CCR4 antagonist is an antibody.
 18. The method ofclaim 17, wherein the antibody binds to CCR4.
 19. The method of claim16, wherein the administration provides for a prolonged localizedconcentration of the CCR4 antagonist.
 20. The method of claim 19,wherein the localized concentration of the CCR4 antagonist is vascular.